Fluorescent probe for zinc

ABSTRACT

A compound represented by general formula (IA) or a salt thereof useful as a fluorescent probe for zinc: wherein R 1  and R 2  represent a hydrogen atom or a group represented by formula (A), wherein X 1 , X 2 , X 3 , and X 4  represent a hydrogen atom, an alkyl group, a 2-pyridylmethyl group, or a protective group for an amino group, and m and n represent 0 or 1, provided that R 1  and R 2  do not simultaneously represent hydrogen atoms; R 3  and R 4  represent a hydrogen atom or a halogen atom; and R 5  and R 6  represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxymethyl group, and R 7  represents a hydrogen atom or an alkyl group

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 10/203,658,filed Feb. 10, 2003, now U.S. Pat. No. 6,903,226, which is a NationalStage Application of International Application No. PCT/JP01/01503, filedFeb. 28, 2001, which was not published in English under PCT Article21(2), entering the National Stage on Aug. 27, 2002, and which claimspriority of Japanese Application No. 2000-50869, filed Feb. 28, 2000.The entire disclosure of application Ser. No. 10/203,658 is consideredas being part of this application, and the entire disclosure ofapplication Ser. No. 10/203,658 is expressly incorporated by referenceherein in its entirety.

TECHNICAL FIELD

The present invention relates to a fluorescent probe for zinc that emitsfluorescence by specifically trapping a zinc ion.

BACKGROUND ART

Zinc is an essential metallic element that is present in the human bodyin the largest amount next to iron. Most zinc ions in cells stronglycouple to proteins and are involved in the maintenance of structure orin the expression of function of the protein. Various reports have beenalso made on the physiological role of free zinc ions, which are presentin the cell in a very small quantity (generally at a level of μM orlower). In particular, zinc ions are considered to be significantlyinvolved in one type of cell death, i.e., apoptosis, and it is reportedthat zinc ions accelerate senile plaque formation in Alzheimer'sdisease.

A compound (a fluorescent probe for zinc), which specifically traps azinc ion to form a complex and emits fluorescence upon the formation ofthe complex, has been conventionally used to measure zinc ions intissue. For example, TSQ (Reyes, J. G., et al., Biol. Res., 27, 49,1994), Zinquin ethyl ester (Tsuda, M. et al., Neurosci., 17, 6678,1997), Dansylaminoethylcyclen (Koike, T. et al., J. Am. Chem. Soc., 118,12686, 1996), and Newport Green (a catalog of Molecular Probe: “Handbookof Fluorescent Probes and Research Chemicals” 6th Edition by Richard P.Haugland pp. 531–540) have been used practically as fluorescent probesfor zinc.

The measurement using TSQ, Zinquin, or Dansylaminoethylcyclen, however,requires the use of a short wavelength excitation light (an excitationwavelength of 367 nm, 368 nm, and 323 nm, respectively). Accordingly,when these fluorescent probes for zinc are used for measurement inliving systems, the short wavelength excitation light may cause damagesof cells (Saibou Kougaku (Cell Technology), 17, pp. 584–595, 1998). Aproblem also arises that the measurement may be readily influenced byautofluorescence generated from cell systems, per se (fluorescenceemitted by NADH or flavin). Further, Dansylaminoethylcyclen has adrawback in that the fluorescence intensity is significantly varieddepending on different environments in which the agent exists at thetime of measurement, e.g., differences in environments such as a type ofa solvent, or extracellular, intracellular, or intramembrane watersolubility or lipophilicity or the like (Tanpakushitsu Kakusan Kouso(Protein, Nucleic Acid and Enzyme), extra number, 42, pp. 171–176,1997). TSQ has a problem in that even distribution in the whole cell isdifficult due to its high lipophilicity. Newport Green has low affinityfor zinc ions and fails to achieve practical measurement sensitivity,although the agent enables measurement with a long wavelength excitationlight. Therefore, the development of a fluorescent probe for zinc hasbeen desired that can measure zinc ions with high sensitivity withoutdamaging cells.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a compound or a saltthereof which can be used as a highly sensitive fluorescent probe forzinc. More specifically, the object of the present invention is toprovide a compound usable as a fluorescent probe for zinc, which canspecifically trap zinc ions and has an excellent fluorescence intensityof a complex after the trap, and which can measure fluorescence with along wavelength excitation light. Another object of the presentinvention is to provide a fluorescent probe for zinc comprising acompound having the above characteristics and a method for measuringzinc ions by using said fluorescent probe for zinc.

The inventors of the present invention have conducted various studies toachieve the foregoing objects. As a result, they found that a compoundhaving a cyclic amine or a polyamine as a substituent has highspecificity with zinc ions, and by trapping zinc ions, the compoundforms a complex which emits strong fluorescence with a excitation lightin longer wavelength range (Japanese Patent Application No. (Hei)11-40325). The inventors have further conducted studies and found that acompounds represented by general formula (I) can form a complex withzinc very rapidly and can emit strong fluorescence. They also found thatthe compounds represented by general formula (I) can react with zincions for a split second in the living organism to form a fluorescentcomplex when they are used as a fluorescent probe for zinc, thereby zincin the living organism can be measured with very high accuracy andsensitivity. The present invention was achieved on the basis of thesefindings.

The present invention thus provides a compound represented by generalformula (IA) or (IB) or a salt thereof:

wherein R¹ and R² independently represent a hydrogen atom or a grouprepresented by formula (A):

wherein X¹, X², X³, and X⁴ independently represent a hydrogen atom, analkyl group, a 2-pyridylmethyl group, or a protective group for an aminogroup, and m and n independently represent 0 or 1, provided that R¹ andR² do not simultaneously represent hydrogen atoms; R³ and R⁴independently represent a hydrogen atom or a halogen atom; R⁵ and R⁶independently represent a hydrogen atom, an alkylcarbonyl group, or analkylcarbonyloxymethyl group, and R⁷ represents a hydrogen atom or analkyl group.

As a preferred embodiment of the present invention, provided is acompound represented by general formula (II) or a salt thereof:

wherein R¹³ and R¹⁴ independently represent a hydrogen atom or a halogenatom; R¹⁷ represents a hydrogen atom or an alkyl group; and R¹⁸represents a hydrogen atom or a protective group for an amino group.According to a preferred embodiment of the aforementioned invention,provided is the aforementioned compound or a salt thereof in which R¹⁷and R¹⁸ independently represent hydrogen atoms. According to morepreferred embodiment, provided is the aforementioned compound or a saltthereof in which a substituted amino group on the benzene ring binds inm-position or p-position relative to the group represented by —COOR¹⁷.

Further, the present invention provides a compound represented bygeneral formula (IIIA) or (IIIB) or a salt thereof:

wherein R²¹ and R²² independently represent a hydrogen atom or a grouprepresented by formula (B):

wherein X¹¹, X¹², X¹³, and X¹⁴ independently represent a hydrogen atom,an alkyl group, a 2-pyridylmethyl group, or a protective group for anamino group, and p and q are independently 0 or 1, provided that R²¹ andR²² do not simultaneously represent hydrogen atoms; Y represents —CO—NH—or —NH—CO—; R²³ and R²⁴ independently represent a hydrogen atom or ahalogen atom; R²⁵ and R²⁶ independently represent a hydrogen atom, analkylcarbonyl group, or an alkylcarbonyloxymethyl group; and R²⁷represents a hydrogen atom or an alkyl group. According to a preferredembodiment of the invention, provides is the aforementioned compound inwhich Y on the benzene ring binds in m-position relative to the grouprepresented by —COOR²⁷ (the corresponding carbonyl group when a lactonering is formed).

From another aspect, the present invention provides a fluorescent probefor zinc which comprises a compound represented by the general formulas(I), (II), or (III) (excluding the compound wherein a protective groupfor an amino group is introduced) or a salt thereof; and a zinc complexconstituted by a compound represented by the general formula (I), (II),or (III) (excluding the compound wherein a protective group for an aminogroup is introduced) or a salt thereof together with a zinc ion. Theaforementioned fluorescent probe for zinc can be used for measuring zincions in tissues or cells.

From further aspect of the present invention, provided are a method formeasuring zinc ions wherein a compound represented by the generalformula (I), (II), or (III) (excluding the compound wherein a protectivegroup for an amino group is introduced) or a salt thereof is used as afluorescent probe for zinc; a method for measuring zinc ions whichcomprises the steps of: (a) reacting a compound represented by thegeneral formula (I), (II), or (III) (excluding the compound wherein aprotective group for an amino group is introduced) or a salt thereofwith zinc ions; and (b) measuring fluorescence intensity of the zinccomplex produced in the above step; and the use of a compoundrepresented by the general formula (I), (II), or (III) (excluding thecompound wherein a protective group for an amino group is introduced) ora salt thereof as a fluorescent probe for zinc.

The present invention is also directed to a compound represented bygeneral formula (IA) or a salt thereof:

wherein R¹ and R² independently represent a hydrogen atom or a grouprepresented by formula (A):

wherein X¹, X², X³, and X⁴ independently represent a hydrogen atom, analkyl group, a 2-pyridylmethyl group, or a protective group for an aminogroup, and m and n independently represent 0 or 1 and at least one of mand n is 1, provided that R¹ and R² do not simultaneously representhydrogen atoms; R³ and R⁴ independently represent a hydrogen atom or ahalogen atom; and R⁵ and R⁶ independently represent a hydrogen atom, analkylcarbonyl group, or an alkylcarbonyloxymethyl group.

The present invention is also directed to a compound represented bygeneral formula (IIIA) or a salt thereof:

wherein R²¹ and R²² independently represent a hydrogen atom or a grouprepresented by formula (B):

wherein X¹¹, X¹², X¹³, and X¹⁴ independently represent a hydrogen atom,an alkyl group, a 2-pyridylmethyl group, or a protective group for anamino group, and p and q are independently 0 or 1 and at least one of pand q is 1, provided that R²¹ and R²² do not simultaneously representhydrogen atoms; Y represents —CO—NH— or —NH—CO—; R²³ and R²⁴independently represent a hydrogen atom or a halogen atom; and R²⁵ andR²⁶ independently represent a hydrogen atom, an alkylcarbonyl group, oran alkylcarbonyloxymethyl group.

The compound represented by the general formula (I), (II), or (III)(limited to the compound wherein a protective group for an amino groupis introduced) is useful as a synthetic intermediate for theaforementioned fluorescent probe for zinc.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows that the fluorescent probe for zinc according to thepresent invention (Compound 6) has excellent zinc ion selectivity.

FIG. 2 shows that the fluorescent probe for zinc according to thepresent invention (Compound 12) has excellent zinc ion selectivity.

FIG. 3 shows that the fluorescent probe for zinc according to thepresent invention (Compound 21) has excellent zinc ion selectivity.

FIG. 4 shows results of a comparison of changes with time influorescence intensity between the fluorescent probes for zinc accordingto the present invention (Compound 6 and Compound 12) and ACF-1 having acyclic polyamine moiety.

FIG. 5 shows a correlation between fluorescence intensity of thefluorescent probes for zinc according to the present invention (Compound6 and Compound 12) and zinc ion concentration.

FIG. 6 shows changes in fluorescence intensity of Compound 12, Compound21, and zinc complexes thereof with relation to pH changes.

FIG. 7 shows a result of investigation on changes in fluorescenceintensity by ischemic stimulus using a rat hippocampal slice.

FIG. 8 shows a result of investigation on changes in fluorescenceintensity in each of regions by ischemic stimulus using a rathippocampal slice.

BEST MODE FOR CARRYING OUT THE INVENTION

All the disclosures in the specification and claims of Japanese PatentApplication No. 2000-50869 are incorporated herein by reference.

“An alkyl group” or an alkyl moiety of a substituent containing thealkyl moiety (for example, an alkylcarbonyl group or analkylcarbonyloxymethyl group) used in the specification means, forexample, a linear, branched, or cyclic alkyl group, or an alkyl groupcomprising a combination thereof having 1 to 12 carbon atoms, preferably1 to 6 carbon atoms, and more preferably 1 to 4 carbon atoms. Morespecifically, a lower alkyl group (an alkyl group having 1 to 6 carbonatoms) is preferred as an alkyl group. Examples of the lower alkylgroups include methyl group, ethyl grouop, n-propyl group, isopropylgroup, cyclopropyl group, n-butyl group, sec-butyl group, isobutylgroup, tert-butyl group, cyclopropylmethyl group, n-pentyl group, andn-hexyl group. When “a halogen atom” is referred to, the term means anyof a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom,and preferably means a fluorine atom, a chlorine atom, or a bromineatom.

Types of protective groups for amino groups are not particularlylimited. For example, a p-nitrobenzenesulfonic acid group, atrifluoroacetyl group, and a trialkylsilyl group can be suitably used.As for the protective groups for amino groups, reference can be made to,for example, “Protective Groups in Organic Synthesis,” (T. W. Greene,John Wiley & Sons, Inc. (1981)).

In the general formulas (IA) and (IB), the positions of R¹ and R²substituted on the benzene ring are not particularly limited. When R² isa hydrogen atom, R¹ may preferably bind in meta-position orpara-position relative to the group represented by —COOR⁷ (orcorresponding carbonyl group when a lactone ring is formed). Theposition of an amino group substituted on the benzene ring in generalformula (II) is not particularly limited. Meta-Position or para-positionrelative to the group represented by —COOR¹⁷ is preferred. In thegeneral formulas (IIIA) and (IIIB), the position of Y substituted on thebenzene ring is not particularly limited. Y may preferably bind inmeta-position relative to the group represented by —COOR²⁷ (orcorresponding carbonyl group when a lactone ring is formed).

In the compounds represented by the general formulas (IA) and (IB),either of R¹ and R² is preferably a hydrogen atom and the other ispreferably a group represented by formula (A). In the group representedby the formula (A), from X¹ to X⁴, preferably X¹ and X², independentlyrepresent a 2-pyridylmethyl group. In the compounds represented by thegeneral formulas (IA) and (IB), preferably, m is 0, n is 1, and X⁴ is ahydrogen atom. In the above particular compounds, both of X¹ and X² arepreferably 2-pyridylmethyl groups. R⁵ and R⁶ are preferably hydrogenatoms, and R⁵ and R⁶ are preferably acetyl groups or acetoxymethylgroups for imaging application. It is preferred that both of R³ and R⁴are hydrogen atoms or chlorine atoms. R⁷ is preferably a hydrogen atom.

In the compound represented by general formula (II), both of R¹³ and R¹⁴are preferably hydrogen atoms or chlorine atoms. R¹⁷ and R¹⁸ arepreferably hydrogen atoms.

In the compounds represented by the general formulas (IIIA) and (IIIB),either of R²¹ and R²² is preferably a hydrogen atom and the other ispreferably a group represented by formula (B). In the group representedby the formula (B), from X¹¹ to X¹⁴, preferably X¹¹ and X¹²,independently represent a 2-pyridylmethyl group. In the compoundsrepresented by the general formulas (IIIA) and (IIIB), preferably, p is0, q is 1, and X¹⁴ is a hydrogen atom. In the above particularcompounds, both of X¹¹ and X¹² are preferably 2-pyridylmethyl groups.Both of R²³ and R²⁴ are preferably hydrogen atoms or chlorine atoms. R²⁵and R²⁶ are preferably hydrogen atoms, and R²⁵ and R²⁶ are preferablyacetyl groups or acetoxymethyl groups for imaging application. R²⁷ ispreferably a hydrogen atom.

The compounds of the present invention represented by the generalformulas (I) to (III) can exist as acid addition salts or base additionsalts. Examples of the acid addition salts include: mineral acid saltssuch as hydrochloride, sulfate, and nitrate; and organic acid salts suchas methanesulfonate, p-toluenesulfonate, oxalate, citrate, and tartrate.Examples of the base addition salts include: metal salts such as sodiumsalts, potassium salts, calcium salts, and magnesium salts; ammoniumsalts; and organic amine salts such as triethylamine salts. In addition,salts of amino acids such as glycine may be formed. The compounds orsalts thereof according to the present invention may exist as hydratesor solvates, and these substances fall within the scope of the presentinvention.

The compounds of the present invention represented by general formulas(IA), (IB), (II), (IIIA), and (IIIB) may have one or more asymmetriccarbons depending on the types of the substituents. Stereoisomers suchas optically active substances based on one or more asymmetric carbonsand diastereoisomers based on two or more asymmetric carbons, as well asany mixtures of the stereoisomers, racemates and the like fall withinthe scope of the present invention. When R⁷, R¹⁷, or R²⁷ is a hydrogenatom, a carboxyl group may form a lactone, and such structural isomersalso fall within the scope of the present invention. A compoundrepresented by general formula (IA) in which R⁵ is a hydrogen atom and acompound represented by general formula (IB) in which R⁷ is a hydrogenatom are tautomers, and similarly, a compound represented by generalformula (IIIA) in which R²⁵ is a hydrogen atom and a compoundrepresented by general formula (IIIB) in which R²⁷ is a hydrogen atomare tautomers. One of ordinary skill in the art would readily recognizethe existence of the tautomers as explained above, and therefore, itshould be understood that any of these tautomers fall within the scopeof the present invention.

Methods for preparing typical compounds of the present invention areshown in the following schemes. The preparation methods shown in theschemes are more specifically detailed in the examples of thespecification. Accordingly, one or ordinary skill in the art can prepareany of the compounds according to the present invention represented bythe general formulas by suitably choosing starting reaction materials,reaction conditions, reagents and the like based on these explanations,and optionally modifying and altering these methods. 4-Aminofluorescein,5-aminofluorescein, and 6-aminofluorescein, which can be used asstarting compounds, can be prepared by methods described in, forexample, “Yuuki Gousei Kagaku (Synthetic Organic Chemistry) IX,”(Tetsuji Kametani, Nankodo Co., Ltd., p. 215 (1977)).

The compound represented by the general formula (III) can be preparedby, for example, a method shown in the following scheme by using acommercially available compound and the like as a reagent and a startingreaction material.

The compounds of the present invention represented by the generalformulas (I), (II), and (III) (excluding a compound having a protectivegroup for an amino group) or salts thereof are useful as fluorescentprobes for zinc. The compounds of the present invention represented bythe general formulas (I), (II), or (III) or salts thereof, per se, donot emit strong fluorescence, whilst they come to emit strongfluorescence after the formation of zinc complexes by trapping zincions. The above compounds or salts thereof are featured that they canspecifically trap zinc ions and form the complex very rapidly. Inaddition, the formed zinc complexes is featured to emit strongfluorescence under a long wavelength excitation light which does notcause any damage to living tissues or cells. Accordingly, the compoundsof the present invention represented by the general formula (I), (II),or (III) or salts thereof are very useful as a fluorescent probes forzinc for measurement of zinc ions in living cells or living tissuesunder a physiological condition. The term “measurement” used in thespecification should be construed in its broadest sense, includingquantitative and qualitative measurement.

The method for using the fluorescent probe for zinc according to thepresent invention is not particularly limited, and the probe can be usedin the same manner as conventional zinc probes. In general, a substanceselected from the group consisting of the compounds represented by thegeneral formula (I) and salts thereof is dissolved in an aqueous mediumsuch as physiological saline or a buffered solution, or in a mixture ofthe aqueous medium and a water-miscible organic solvent such as ethanol,acetone, ethylene glycol, dimethylsulfoxide, and dimethylformamide, andthen the resultant solution is added to a suitable buffered solutioncontaining cells or tissues and a fluorescence spectrum can be measured.

For example, the zinc complexes of Compound 6 and Compound 12 shown inthe above scheme have the excitation wavelengths of 491 nm and 492 nm,and the fluorescence wavelengths of 513 nm and 514 nm, respectively.When the compound is used at a concentration of about 1 to 10 μM, zincions with a concentration of 10 μM or below can be measured. Thefluorescent probe for zinc according to the present invention may becombined with a suitable additive to use in the form of a composition.For example, the fluorescent probe for zinc can be combined withadditives such as a buffering agent, a solubilizing agent, and a pHmodifier.

Compound 22, for example, has lipophilicity such a degree that it caneasily permeate cell membranes. After Compound 22 permeates cellmembranes, the compound is hydrolyzed by an esterase present in thecytoplasm, thereby Compound 12 is produced. Compound 12 can hardlypermeate cell membranes due to its water-solubility, and for thisreason, Compound 12 can be retained intracellularly for a prolongedperiod of time. Accordingly, Compound 22 is very useful for measurementof zinc ions existing in an individual cell.

EXAMPLES

The present invention will be more specifically explained with referenceto the following examples. However, the scope of the present inventionis not limited to these examples. The compound numbers in the examplescorrespond to those used in the above schemes.

Example 1

Synthesis of Compound 6

Cesium carbonate (5.2 g, 16 mmol) was added to a solution of4-aminofluorescein (1) (2.5 g, 7.2 mmol) dissolved in 50 ml ofdimethylformamide. Subsequently, pivaloyl anhydride (3.1 ml, 15 mmol)was added to this solution, and the mixture was stirred at roomtemperature for 1 hour. The reaction solution was filtered using aKiriyama funnel, and dimethylformamide was evaporated under reducedpressure. The residue was purified by column chromatography on silicagel to obtain 3.6 g of Compound 2 (white solid, yield: 97%).

¹H-NMR (CDCl₃, 300 MHz): 7.19 (m, 1H), 7.02 (d, 2H, J=2.4), 6.93–6.94(m, 2H), 6.88 (d, 2H, J=8.7), 6.77 (dd, 2H, J=8.7, 2.4), 4.06 (br, 2H),1.34 (s, 18H)

MS (FAB): 516 (M⁺+1)

m.p.: 206–208° C. (recrystallized from methanol)

Compound 2 (1.0 g, 2.0 mmol) was dissolved in 15 ml of pyridine and thesolution was added with 4-nitrobenzenesulfonyl chloride (1.2 g, 5.3mmol), and the mixture was then stirred at room temperature for 6 hours.Pyridine was evaporated under reduced pressure, and the residue wasdissolved in 25 ml of ethyl acetate. The ethyl acetate solution waswashed with 2N hydrochloric acid and saturated brine, and was then driedover sodium sulfate. After ethyl acetate was evaporated under reducedpressure, purification was carried out by column chromatography onsilica gel to obtain 1.2 g of Compound 3 (white solid, yield: 88%).

¹H-NMR (CDCl₃, 300 MHz): 8.33 (d, 2H, J=9.0), 8.05 (d, 2H, J=9.0), 7.69(d, 1H, J=2.2), 7.45 (dd, 1H, J=8.2, 2.2), 7.07 (d, 1H, J=8.2),7.06–7.04 (m, 2H), 6.77–6.74 (m, 4H), 1.36 (s, 18H)

MS (FAB): 701(M⁺+1)

m.p.: 245–247° C. (recrystallized from ethyl acetate+n-hexane)

Cesium carbonate (0.48 g, 1.5 mmol) and 1,2-dibromoethane (1.3 ml, 14mmol) were added to absolution of Compound 3 (0.97 g, 1.4 mmol)dissolved in 25 ml of dimethylformamide, and the mixture was stirred at60° C. for 20 hours. Dimethylformamide was evaporated under reducedpressure and dissolved in 50 ml of ethyl acetate. The ethyl acetatesolution was washed with water and saturated brine, and then dried oversodium sulfate. After ethyl acetate was evaporated under reducedpressure, purification was carried out by column chromatography onsilica gel to obtain 0.78 g of Compound 4 (white solid, yield: 70%).

¹H-NMR (CDCl₃, 300 MHz): 8.38 (d, 2H, J=9.0), 7.86 (d, 2H, J=9.0), 7.76(d, 1H, J=2.0), 7.45 (dd, 1H, J=8.0, 2.0), 7.17 (d, 1H, J=8.0), 7.08 (m,2H), 6.85–6.84 (m, 4H), 4.01 (t, 2H, J=6.8), 3.45 (t, 2H, J=6.8), 1.37(s, 18H)

MS (FAB): 807, 809 (M⁺+1)

m.p.: 280–281° C. (recrystallized from acetonitrile)

Compound 4 (0.10 g, 0.13 mmol) was suspended in 4 ml of acetonitrile,and the suspension was added with potassium iodide (55 mg, 0.33 mmol),potassium carbonate (43 mg, 0.31 mmol), and 2,2′-dipicolylamine (78 mg,0.39 mmol), and then the mixture was refluxed for 14 hours. Afteracetonitrile was evaporated under reduced pressure, the product wasdissolved in an aqueous solution of 2N sodium carbonate, followed byextraction with methylene chloride. The methylene chloride layer waswashed with saturated brine and was then dried over sodium sulfate.Methylene chloride was evaporated under reduced pressure, followed bypurification by column chromatography on silica gel to obtain 80 mg ofCompound 5 (light yellow oil, yield: 69%).

¹H-NMR (CDCl₃, 300 MHz): 8.47–8.45 (m, 2H), 8.32 (d, 2H, J=9.0), 7.77(d, 2H, J=9.0), 7.69–7.61 (m, 3H), 7.61 (d, 2H, J=7.9), 7.27–7.23 (m,1H), 7.14 (m, 2H), 7.07 (d, 2H, J=2.2), 6.99 (d, 1H, J=8.0), 6.82 (dd,2H, J=8.6, 2.2), 6.72 (d, 2H, J=8.6) 3.82 (s, 4H), 3.82 (m, 2H), 2.72(t, 2H, J=6.4), 1.37 (s, 18H)

MS (FAB): 926 (M⁺+1)

Potassium carbonate (26 mg, 0.19 mmol) and thiophenol (12 μl, 0.12 mmol)were added to a solution of Compound 5 (34 mg, 37 μmol) dissolved in 4ml of dimethylformamide, and the mixture was stirred at room temperaturefor 3 hours. A solution of potassium hydroxide (70 mg, 1.2 mmol),dissolved in 1 ml of methanol and 1 ml of water, was added to thereaction mixture, and the mixture was stirred at room temperature for 20hours. After 2 ml of 2N hydrochloric acid was added to the mixture, thesolvent was evaporated under reduced pressure. The product was suspendedin 10 ml of ethanol and filtered, and then the ethanol was evaporatedunder reduced pressure. The residue was purified by reversed-phase HPLCto obtain 15 mg of Compound 6 (brown solid, yield: 70%).

¹H-NMR (CD₃OD, 300 MHz): 8.61–8.59 (m, 2H), 8.04–7.98 (m, 2H), 7.63 (d,2H, J=7.9), 7.51–7.46 (m, 2H), 7.14 (d, 1H, J=2.0), 7.02 (d, 2H, J=9.0),6.95–6.87 (m, 4H), 6.79 (dd, 2H, J=9.0, 2.4), 4.46 (s, 4H), 3.50 (t, 2H,J=6.0), 3.25 (m, 2H)

MS (FAB): 573 (M⁺+1)

Example 2

Synthesis of Compound 12

Compound 8 (4.4 g) was obtained from 5-aminofluorescein (7) (3.5 g, 10mmol) in the same manner as that of the synthesis of Compound 2 (whitesolid, yield: 84%).

¹H-NMR (CDCl₃, 300 MHz): 7.77 (d, 1H, J=7.9), 7.01 (d, 2H, J=2.0), 6.95(d, 2H, J=8.6), 6.80–6.75 (m, 3H), 6.22 (d, 1H, J=1.7), 4.21 (br, 2H),1.36 (s, 18H)

MS (FAB): 516 (M⁺+1)

m.p.: 161–163° C. (recrystallized from methanol)

Compound 9 (4.1 g) was obtained from Compound 8 (3.6 g, 6.9 mmol) in thesame manner as that of the synthesis of Compound 3 (white solid, yield:84%).

¹H-NMR (CDCl₃, 300 MHz): 8.61 (br, 1H), 8.20 (d, 2H, J=9.0), 7.88 (d,1H, J=8.3), 7.81 (d,.2H, J=9.0), 7.33–7.29 (m, 1H), 7.05 (d, 2H, J=2.2),6.84 (d, 1H, J=1.8), 6.74 (dd, 2H, J=8.6, 2.2), 6.69 (d, 2H, J=8.6),1.38 (s, 18H)

MS (FAB): 701 (M⁺+1)

m.p.: 189–191° C. (recrystallized from ethyl acetate+n-hexane)

Compound 10 (0.35 g) was obtained from Compound 9 (0.51 g, 9.73 mmol) inthe same manner as that of the synthesis of Compound 4 (white solid,yield: 60%).

¹H-NMR (CDCl₃, 300 MHz): 8.11 (d, 2H, J=9.0), 8.10–8.09 (m, 1H), 7.71(dd, 1H, J=8.2, 1.8), 7.56 (d, 2H, J=9.0), 7.02 (d, 2H, J=2.2), 6.86(dd, 2H, J=8.6, 2.2), 6.79 (d, 2H, J=8.6), 6.43 (d, 1H, J=1.8), 3.85 (t,2H, J=6.6), 3.40 (t, 2H, J=6.6), 1.38 (s, 18H)

MS (FAB): 807, 809 (M⁺+1)

m.p.: 268–269° C. (recrystallized from acetonitrile)

Compound 11 (0.27 g) was obtained from Compound 10 (0.31 g, 0.38 mmol)in the same manner as that of the synthesis of Compound 5 (light yellowsolid, yield: 75%).

¹H-NMR (CDCl₃, 300 MHz): 8.45–8.42 (m, 2H), 8.06 (d, 2H, J=9.0), 7.96(d, 1H, J=8.3), 7.64–7.59 (m, 2H), 7.52 (d, 2H, J=9.0), 7.53–7.50 (m,1H), 7.33 (d, 2H, J=7.7), 7.17 (m, 2H), 7.00 (d, 2H, J=2.2), 6.78 (dd,2H, J=8.6, 2.2), 6.64 (d, 2H, J=8.6), 6.48 (d, 1H, J=1.3), 3.71 (s, 4H),3.67 (t, 2H, J=6.2), 2.67 (t, 2H, J=6.2), 1.37 (s, 18H)

MS (FAB): 926 (M⁺+1)

m.p.: 146–148° C. (recrystallized from methanol)

Compound 12 (6.6 mg) was obtained from Compound 11 (20 mg, 22 μmol) inthe same manner as that of the synthesis of Compound 6 (brown solid,yield: 53%).

¹H-NMR (CD₃OD, 300 MHz): 8.44–8.42 (m, 2H), 7.94–7.88 (m, 2H), 7.60 (d,1H, J=8.4), 7.49 (d, 2H, J=7.9), 7.45–7.41 (m, 2H), 6.71 (br, 1H), 6.65(d, 2H, J=2.4), 6.61 (d, 2H, J=8.8), 6.51 (dd, 2H, J=8.8, 2.4), 6.02 (d,1H, J=1.8), 4.30 (s, 4H), 3.28 (t, 2H, J=6.0), 3.03 (t, 2H, J=6.0)

MS (FAB): 573 (M⁺+1)

Example 3

Synthesis of Compound 15

4-Nitrophthalic anhydride (13) (16 g, 84 mmol) and 4-chlororesorcinol(14) (24 g, 0.17 mol) were dissolved in 250 ml of methanesulfonic acid,and the mixture was stirred under argon at 80° C. for 60 hours. Themixture was cooled to room temperature and then added in small portionsto 1.4 L of ice water. The precipitated solid was collected byfiltration to obtain 37 g of Compound 15 (quantitative yield).

Synthesis of Compound 16

Compound (15) (20 g, 45 mmol) was suspended in 700 ml of water, and thesuspension was added with sodium sulfate nonahydrate (54 g, 0.23 mol)and sodium hydrosulfide n-hydrate (20 g, 0.25 mol, about 70% of sodiumhydrosulfide), and the mixture was refluxed under argon for 20 hours.After cooled to room temperature, the mixture was added withhydrochloric acid to adjust pH at 3 to 4. The precipitated solid wascollected by filtration to obtain 19 g of Compound (16) (quantitativeyield).

Synthesis of Compound 17

Compound (17) (3.9 g) was obtained from Compound (16) (4.4 g, 11 mmol)in the same manner as that of the synthesis of Compound (2) (yield:62%).

MS (FAB): 584, 586, 588 (M⁺+1)

Synthesis of Compounds 18 and 18′

Compound (18) (1.9 g) and 1.8 g of Compound (18′) were obtained fromCompound (17) (3.8 g, 6.5 mmol) in the same manner as that of thesynthesis of Compound (3) (yield: 38% for Compound (18); 35% forCompound (18′)).

Compound (18):

¹H NMR (CDCl₃, 300 MHz): 8.38 (d, 2H, J=8.7), 8.07 (d, 2H, J=8.7), 7.72(d, 1H, J=2.1), 7.48 (dd, 1H, J=8.1, 2.1), 7.12 (d, 1H, J=8.1), 7.11 (s,2H), 6.77 (s, 2H), 1.40 (s, 18H)

MS (FAB): 769, 771, 773 (M⁺+1)

Compound (18′):

¹H-NMR (CDCl₃, 300 MHz): 8.26 (d, 2H, J=8.6), 7.93 (d, 1H, J=8.4), 7.84(d, 2H, J=8.6), 7.27 (dd, 1H, J=8.4, 2.0), 7.13 (s, 2H), 6.99 (d, 1H,J=2.0), 6.75 (s, 2H), 1.42 (s, 18H)

MS (FAB): 769, 771, 773 (M⁺+1)

Synthesis of Compound 19

Compound (19) (1.2 g) was obtained from Compound (18) (1.5 g, 2.0 mmol)in the same manner as that of the synthesis of Compound (4) (yield:66%).

¹H-NMR (CDCl₃, 300 MHz): 8.39 (d, 2H, J=9.0), 7.85 (d, 2H, J=9.0), 7.79(d, 1H, J=2.0), 7.51 (dd, 1H, J=8.2, 2.0), 7.18 (d, 1H, J=8.2), 7.14 (s,2H), 6.89 (s, 2H), 4.06 (t, 2H, J=6.8), 3.50 (t, 2H, J=6.8), 1.40 (s,18H)

MS (FAB): 875, 877, 879, 881 (M⁺+1)

Synthesis of Compound 19′

Compound (19′) (0.70 g) was obtained from Compound (18′) (1.5 g, 2.0mmol) in the same manner as that of the synthesis of Compound (4)(yield: 40%).

¹H-NMR (CDCl₃, 300 MHz): 8.19 (d, 2H, J=9.0), 8.13 (d, 1H, J=8.3), 7.70(brd, 1H), 7.62 (d, 2H, J=9.0), 7.11 (s, 2H), 6.84 (s, 2H), 6.63 (d, 1H,J=1.8), 3.94 (t, 2H, J=6.4), 3.46 (t, 2H, J=6.4), 1.41 (s, 18H)

MS (FAB): 875, 877, 879, 881 (M⁺+1)

Synthesis of Compound 20

Compound (20) (0.56 g) was obtained from Compound (19) (1.0 g, 1.1 mmol)in the same manner as that of the synthesis of Compound (5) (yield:49%).

¹H-NMR (CDCl₃, 300 MHz): 8.50–8.47 (m, 2H), 8.33 (d, 2H, J=8.7), 7.76(d, 2H, J=8.7), 7.70–7.60 (m, 3H), 7.46 (d, 2H, J=7.9), 7.32 (brd, 1H,J=8.3), 7.16–7.12 (m, 2H), 7.14 (s, 2H), 7.00 (d, 1H, J=8.3), 6.79 (s,2H), 3.87 (t, 2H, J=6.0), 3.83 (s, 4H), 2.76 (t, 2H, J=6.0), 1.41 (s,18H)

MS (FAB): 994, 996, 998 (M⁺+1)

Synthesis of Compound 20′

Compound (20′) (75 mg) was obtained from Compound (19′) (0.20 g, 0.23mmol) in the same manner as that of the synthesis of Compound (5)(yield: 33%).

¹H-NMR (CDCl₃, 300 MHz): 8.43–8.41 (m, 2H), 8.17 (d, 2H, J=9.0), 7.97(d, 1H, J=8.3), 7.63–7.57 (m, 2H), 7.56 (d, 2H, J=9.0), 7.49 (brd, 1H,J=8.3), 7.31 (d, 2H, J=7.7), 7.16–7.12 (m, 2H), 7.08 (s, 2H), 6.79 (s,2H), 6.72 (d, 2H, J=1.1), 3.74 (t, 2H, J=6.2), 3.71 (s, 4H), 2.74 (t,2H, J=6.2), 1.40 (s, 18H)

MS (FAB): 994, 996, 998 (M⁺+1)

Synthesis of Compound 21

Compound (21) (98 mg) was obtained from Compound (20) (0.26 g, 0.26mmol) in the same manner as that of the synthesis of Compound (6)(yield: 35%).

¹H-NMR (CD₃OD, 300 MHz): 8.56 (brd, 2H, J=4.8), 7.98–7.91 (m, 2H), 7.57(d, 2H, J=7.9), 7.46–7.41 (m, 2H), 6.94–6.81 (m, 3H), 6.73 (s, 2H), 6.56(s, 2H), 4.48 (s, 4H), 3.50 (t, 2H, J=5.5), 3.29 (t, 2H, J=5.5)

MS (FAB): 641, 643, 645 (M⁺+1)

Synthesis of Compound 21′

Compound (21′) (58 mg) was obtained from Compound (20′) (0.20 g, 0.20mmol) in the same manner as that of the synthesis of Compound (6)(yield: 26%).

¹H-NMR (CD₃OD, 300 MHz): 8.45–8.43 (m, 2H), 7.93–7.88 (m, 2H), 7.58 (d,1H, J=8.6), 7.50 (d, 2H, J=7.9), 7.45–7.41 (m, 2H), 6.72 (s, 2H),6.73–6.88 (m, 1H), 6.58 (s, 2H), 6.01 (d, 1H, J=1.8), 4.30 (s, 4H), 3.27(t, 2H, J=5.7), 3.06 (t, 2H, J=5.7)

MS (FAB): 641, 643, 645 (M⁺+1)

Synthesis of Compound 22

Compound (12) (140 mg, 0.13 mmol) was suspended in 10 ml ofacetonitrile, and the suspension was added with cesium carbonate (0.19g, 0.30 mmol), and then with 28 μl of acetic anhydride portionwise.After the mixture was stirred at room temperature for 1 hour, thereaction mixture was filtered. The solvent was evaporated under reducedpressure, followed by purification by column chromatography on silicagel to obtain 79 mg of Compound (22) (yield: 94%).

¹H-NMR (CDCl₃, 300 MHz): 8.51–8.49 (m, 2H), 7.73 (d, 1H, J=8.4),7.60–7.54 (m, 2H), 7.31 (d, 2H, J=7.7), 7.15–7.11 (m, 2H), 7.05 (d, 2H,J=2.2), 6.96 (d, 2H, J=8.6), 6.80 (dd, 2H, J=2.2, 8.6), 6.77–6.74 (m,1H), 6.48 (br, 1H), 6.02 (d, 1H, J=1.7), 3.86 (s, 4H), 3.06 (br, 2H),2.82 (t, 2H, J=5.1), 2.31 (s, 6H)

Example 4

Compound 6 obtained in Example 1 and Compound 12 obtained in Example 2were used to evaluate selectivity for zinc ions. 5 μM of Compound 6 orCompound 12 was added in 100 mM HEPES buffer (pH 7.5) containing variousmetal ions (5 μM or 5 mM). The fluorescence intensity was measured atthe excitation wavelength of 491 nm and the fluorescence wavelength of513 nm for Compound 6, and the excitation wavelength of 492 nm and thefluorescence wavelength of 514 nm for Compound 12. The results are shownin FIG. 1 (Compound 6) and FIG. 2 (Compound 12). 1 μM of Compound 21 wasadded in 100 mM HEPES buffer (pH 7.5) containing various metal ions (1μM or 5 mM), and the fluorescence intensity was measured at theexcitation wavelength of 505 nm and the fluorescence wavelength of 522nm. The results are shown in FIG. 3

In the figures, the fluorescence intensities on the ordinate axis areshown as numerical values with addition of each metal ion relative tothe fluorescence intensity without addition of metal ion which is takenas 1. It is clearly understood that Compound 6 and Compound 12 of thepresent invention have extremely high selectivity for zinc ions, and thecompounds give absolutely no increase of fluorescence intensity even inthe presence of sodium ions, potassium ions, calcium ions, and magnesiumions at high concentration (5 mM), which exist in a living organism inlarge amounts. It is also clearly understood that these metal ions donot affect the increase in fluorescence intensity by zinc ions.

Compound 21 exhibited high selectivity for zinc. In particular, theaddition of sodium, potassium, calcium, and magnesium at highconcentration (5 mM), which are metal ions present abundant in livingorganisms, gives almost no increase in fluorescence intensity. Thesemetal ions did not affect the increase in fluorescence intensity causedby zinc.

Example 5

Zinc ion (final concentration 5 μM or 50 μM) was added in 100 mM HEPES(pH 7.5) containing 5 μM Compound 6, Compound 12, or ACF-1 (a compoundhaving a cyclic polyamine moiety described as Compound (20) in Example 1in Japanese Patent Application No. (Hei) 11-40325) to measurefluorescence intensity. The fluorescence intensity was measured at theexcitation wavelength of 491 nm and the fluorescence wavelength of 513nm for Compound 6, the excitation wavelength of 492 nm and thefluorescence wavelength of 514 nm for Compound 12, and the excitationwavelength of 495 nm and the fluorescence wavelength of 515 nm forACF-1. The results are shown in FIG. 4. In the figure, the ordinate axisrepresents relative fluorescence intensity. As clearly indicated theresults, the fluorescence intensity is not instantly increased by ACF-1,whilst fluorescence intensities were instantly increased by Compound 6and Compound 12 of the present invention. Accordingly, the use of thecompound according to the present invention enables very quick detectionof zinc, and also enables the detection of rapid change in theconcentration of zinc.

Example 6

Zinc ions at various concentrations were added in 100 mM HEPES buffer(pH 7.5) containing 5 μM Compound 6, Compound 12, ACF-1, or NewportGreen (Handbook of Fluorescent Probes and Research Chemicals, 6thEdition by Richard P. Haugland, pp. 531–540), and changes influorescence intensity were measured. The fluorescence intensity wasmeasured at the excitation wavelength of 491 nm and the fluorescencewavelength of 513 nm for Compound 6, the excitation wavelength of 492 nmand the fluorescence wavelength of 514 nm for Compound 12, theexcitation wavelength of 495 nm and the fluorescence wavelength of 515nm for ACF-1, and the excitation wavelength of 505 nm and thefluorescence wavelength of 530 nm for Newport Green. The results areshown in FIG. 5. In the figure, the fluorescence intensities on theordinate axis are shown as numerical values with addition of each metalion relative to the fluorescence intensity without addition of metal ionwhich is taken as 1. Compound 6 and Compound 12 of the present inventionexhibited a high detection sensitivity. In particular, the detectionsensitivity of Compound 12 was very high, which verifies an optimumcombination of the chelater moiety and the fluorescence-emitting moietyof the compound.

Example 7

Changes in fluorescence intensity of Compound 12, Compound 21, and zinccomplexes thereof were investigated with relation to pH changes. Thefluorescence intensity was measured at the excitation wavelength of 492nm and the fluorescence wavelength of 514 nm for Compound 12, and theexcitation wavelength of 505 nm and the fluorescence wavelength of 522nm for Compound 21. The results are shown in FIG. 6.

Buffers used are as follows.

100 mM Cl₂CHCOOH—Cl₂CHCOONa buffer (pH 2.0)

100 mM ClCH₂COOH—ClCH₂COONa buffer (pH 3.0)

100 mM AcOH—AcONa buffer (pH 4.0, 4.5, 5.0)

100 mM MES buffer (pH 5.5, 6.0, 6.5)

100 mM HEPES buffer (pH 7.0, 7.5, 8.0)

100 mM CHES buffer (pH 8.5)

The fluorescence intensity of Compound 21 was more stable than Compound12 at pH of around 7.4 which is an intracellular pH, which indicatesthat the probe is hardly influenced by intracellular pH changes.

Example 8

The change in fluorescence intensity by ischemic stimulus wasinvestigated using a rat hippocampal slice.

10 μM of Compound 22 was added to a rat hippocampal slice and incubated,and then the slice was subjected to ischemic stimulus for 10 minutes (2to 12 minutes in the drawing) to observe a change with time influorescent images. The results are shown in FIG. 7.

For preparation of the hippocampal slice, Ringer's solutions having thefollowing formulations were used.

(1) Ringer's solution

-   -   Formulation: 124 mM NaCl, 1.25 mM NaH₂PO₄, 2.5 mM KCl, 2 mM        CaCl₂, 26 mM NaHCO₃, 1 mM MgCl₂, 10 mM glucose

(2) Ringer's solution for ischemia

-   -   Formulation: 124 mM NaCl, 1.25 mM NaH₂PO₄, 2.5 mM KCl, 2 mM        CaCl₂, 26 mM NaHCO₃, 1 mM MgCl₂, 10 mM 2-deoxyglucose

(3) Choline-Ringer's solution

-   -   Formulation: 124 mM choline, 1.25 mM NaH₂PO₄, 2.5 mM KCl, 0.5 mM        CaCl₂, 26 mM NaHCO₃, 2.5 mM MgCl₂, 10 mM glucose        The Ringer's solutions used for the preparation and measurement        of the slices were kept under constant bubbling of 95% O₂/5%        CO₂.

A Wistar rat (200 to 250 g, male) was anesthetized with ether. Afterdecapitation, the whole brain was rapidly extirpated and put into theice-cooled choline-Ringer's solution, and allowed to stand for 10minutes. After the brain was cut into left and right hemispheriums onShale loaded with the ice-cooled choline-Ringer's solution and asorbet-like choline-Ringer's solution, the interbrain was removed andthe hippocampus was taken out using a spatula. The hippocampus wasplaced on agar and fixed to the agar with pins, and sliced in the widthof 300 μm using a rotary slicer. The sliced hippocampus was put into theRinger's solution heated to 30° C. and allowed to stand for 30 minutesto 1 hour. The sliced hippocampus was kept in the Ringer's solution atroom temperature until it was put to use.

Subsequently, a 10 mM solution of Compound 22 dissolved in DMSO wasdiluted to 10 μM with the Ringer's solution. The sliced hippocampus, wasput into the resulting solution and incubated under a shaded conditionat room temperature for 1.5 hour. After the sliced hippocampus was putinto the fresh Ringer's solution and washed for about 30 minutes to 1hour and 30 minutes, and then transferred into a chamber and subjectedto measurement. The warmed Ringer's solution was circulated (flow rateof 2 to 3 ml/minute) in the chamber to constantly keep the temperatureat 33 to 34° C. Measurement was carried out using an inverted microscope(Olympus IX-70, objective lens: 4×, dichroic mirror: 505 nm).

Ischemic stimulus was carried out by exchanging the Ringer's solutioncirculated in the chamber in the following manner.

Ringer's solution (95% O₂+5% CO₂ bubbling): 2 minutes (01 00. 00 to 0200. 00 in the figure)→Ringer's solution for ischemia (95% N₂+5% CO₂bubbling): 10 minutes (03 00. 00 to 12 00. 00 in the figure)→Ringer'ssolution (95% O₂+5% CO₂ bubbling): 4 minutes (13 00. 00 to 16 00. 00 inthe figure).

As a result, in the CA1 region, where cell death was reported to becaused by, ischemic stimulus, the fluorescence intensity was found to beincreased about 3 minutes after the initiation of ischemic stimulus.

Further, the fluorescence intensity was most remarkably increased in theCA1 region (1, 2, 3 in the drawing) after ischemic stimulus as shown inFIG. 8. The fluorescence intensity was also increased in the CA3 region(site 4 in the figure) and the dentate gyrus (sites 6 and 7 in thefigure). The ordinate axis of the graph shows fluorescence intensity atthe time of initiation of measurement (0.00 sec) which is taken as 1.00.

INDUSTRIAL APPLICABILITY

The compound of the present invention is useful as a fluorescent probefor measurement of zinc. More specifically, the compound of the presentinvention is characterized to form a complex with zinc very quickly anddetection sensitivity is very high. Accordingly, the compound of thepresent invention is very useful as an agent for accurately measuringrapid changes in concentration of zinc ions in a living organism.

The invention claimed is:
 1. A compound represented by general formula(IA) or a salt thereof:

wherein R¹ and R² independently represent a hydrogen atom or a grouprepresented by formula (A):

wherein X¹, X², X³, and X⁴ independently represent a hydrogen atom, analkyl group, a 2-pyridylmethyl group, or a protective group for an aminogroup, and m and n independently represent 0 or 1 and at least one of mand n is 1, provided that R¹ and R² do not simultaneously representhydrogen atoms; R³ and R⁴ independently represent a hydrogen atom or ahalogen atom; and R⁵ and R⁶ independently represent a hydrogen atom, analkylcarbonyl group, or an alkylcarbonyloxymethyl group.
 2. The compoundaccording to claim 1, wherein one of R¹ and R² is hydrogen, and theother of R¹ and R² is represented by formula (A) wherein X¹ and X² are2-pyridylmethyl groups.
 3. The compound according to claim 2, wherein R⁵and R⁶ are hydrogen atoms.
 4. The compound according to claim 2, whereinR⁵ and R⁶ are acetyl groups or acetoxymethyl groups.
 5. The compoundaccording to claim 1, wherein m is 0, and n is
 1. 6. A compoundrepresented by general formula (IIIA) or a salt thereof:

wherein R²¹ and R²² independently represent a hydrogen atom or a grouprepresented by formula (B):

wherein X¹¹, X¹², X¹³, and X¹⁴ independently represent a hydrogen atom,an alkyl group, a 2-pyridylmethyl group, or a protective group for anamino group, and p and q are independently 0 or 1 and at least one of pand q is 1, provided that R²¹ and R²² do not simultaneously representhydrogen atoms; Y represents —CO—NH— or —NH—CO—; R²³ and R²⁴independently represent a hydrogen atom or a halogen atom; and R²⁵ andR²⁶ independently represent a hydrogen atom, an alkylcarbonyl group, oran alkylcarbonyloxymethyl group.
 7. The compound according to claim 6,wherein one of R²¹ and R²² is hydrogen, and the other of R²¹ and R²² isrepresented by formula (B) wherein X¹¹ and X¹² are 2-pyridylmethylgroups.
 8. The compound according to claim 7, wherein R²⁵ and R²⁶ arehydrogen atoms.
 9. The compound according to claim 7, wherein R²⁵ andR²⁶ are acetyl groups or acetoxymethyl groups.
 10. The compoundaccording to claim 6, wherein p is 0, and q is
 1. 11. A fluorescentprobe for zinc which comprises a compound according to claim 1, providedthat a compound is excluded wherein a protective group for an aminogroup is introduced, or a salt thereof.
 12. A zinc complex which isformed by a compound according to claim 1, provided that a compound isexcluded wherein a protective group for an amino group is introduced, ora salt thereof together with a zinc ion.
 13. A method for measuring azinc ion which comprises: (a) reacting a compound according to claim 1,provided that a compound is excluded wherein a protective group for anamino group is introduced, or a salt thereof with a zinc ion to form azinc complex; and (b) measuring fluorescence intensity of the zinccomplex.
 14. A fluorescent probe for zinc which comprises a compoundaccording to claim 6, provided that a compound is excluded wherein aprotective group for an amino group is introduced, or a salt thereof.15. A zinc complex which is formed by a compound according to claim 6,provided that a compound is excluded wherein a protective group for anamino group is introduced, or a salt thereof together with a zinc ion.16. A method for measuring a zinc ion which comprises: (a) reacting acompound according to claim 6, provided that a compound is excludedwherein a protective group for an amino group is introduced, or a saltthereof with a zinc ion to form a zinc complex; and (b) measuringfluorescence intensity of the zinc complex.
 17. A compound representedby general formula (IA) or a salt thereof:

wherein R¹ and R² independently represent a hydrogen atom or a grouprepresented by formula (A):

wherein X¹, X², X³, and X⁴ independently represent a hydrogen atom, analkyl group, a 2-pyridylmethyl group, or a protective group for an aminogroup, and m and n independently represent 0 or 1; one of R¹ and R² ishydrogen, and the other of R¹ and R² is represented by formula (A)wherein X¹ and X² are 2-pyridylmethyl groups; R³ and R⁴ independentlyrepresent a hydrogen atom or a halogen atom; and R⁵ and R⁶ independentlyrepresent a hydrogen atom, an alkylcarbonyl group, or analkylcarbonyloxymethyl group.
 18. A compound represented by generalformula (IIIA) or a salt thereof:

wherein R²¹ and R²² independently represent a hydrogen atom or a grouprepresented by formula (B):

wherein X¹¹, X¹², X¹³, and X¹⁴ independently represent a hydrogen atom,an alkyl group, a 2-pyridylmethyl group, or a protective group for anamino group, and p and q are independently 0 or 1, provided that R²¹ andR²² do not simultaneously represent hydrogen atoms; Y represents —CO—NH—or —NH—CO—; R²³ and R²⁴ independently represent a hydrogen atom or ahalogen atom; and R²⁵ and R²⁶ are hydrogen atoms.
 19. The compoundaccording to claim 6, wherein R²⁵ and R²⁶ independently represent ahydrogen atom or an alkylcarbonyloxymethyl group.